Platelets play a major role in the development of thrombus formation in patients with arterial thromboembolic diseases. Abnormal platelet behavior is associated with several pathological conditions, such as acute coronary syndrome (“ACS”), and in patients undergoing percutaneous transluminal coronary angioplasty (“PTCA”). Fitzgerald D. J. et al., N. Engl. J. Med. (1986) 315:983-989; Hamm C. W. et al., J. Am. Coll. Cardiol. (1987) 10:998-1006; Schultheiss H. P. et al., Eur. J. Clin. Invest. (1994) 24:243-247; Tschoepe D. et al., Circulation (1993) 88:37-42. Consequently, anticoagulant and anti-platelet therapies are widely used for cardiovascular disease patients. For example, treatment of ACS patients with aspirin, ADP receptor inhibitors, peptide and non-peptide glycoprotein IIb/IIIa (GPIIb/IIIa) receptor inhibitors, and other novel inhibitors of platelet function have been shown to significantly improve both short-term and long-term outcome by reducing the incidence of recurrent MI and death. Lewis H. D. et al., N. Engl. J. Med. (1983) 309:396-403; Jarvis B. and K. Simpson, Drugs (2000) 60: 347-377; Moshfegh K. et al., J. Am. Coll. Cardiol. (2000) 36:699-705; Rupprecht H. J. et al. Circulation (1998) 97:1046-1052; PRISM Study Group, N. Engl. J. Med. (1998) 338:1498-505; PRISM-PLUS Study Group, N. Engl. J. Med. (1998) 338:1488-1997; The EPIC Investigators, N. Engl. J. Med. (1994) 330:956-961; The CAPTURE Investigators, Lancet (1997) 349:1429-1435; Mascelli M. A. et al., Circulation (1998) 97:1680-1688. Inter-individual variation in response to the monoclonal antibody, abciximab, has been reported both in terms of extent and duration of inhibition of platelet function. Mascelli M. A. et al., Circulation (1998) 97:1680-1688; Bihour C. et al., Arterioscler. Thromb. Vasc. Biol. (1999) 16:212-9; Kleiman N. S. et al., J. Am. Coll. Cardiol. (1995) 26:1665-1671. Thus, anti-platelet therapies represent an important component in treatment regimens for numerous vascular pathologies.
Although high levels of platelet function inhibition has been reported to be associated with a decrease in the incidence of major adverse cardiac events, there is a clinical risk of thrombosis or bleeding associated with anti-platelet and anticoagulant treatments. Aguirre F. V. et al., Circulation (1995) 91:2882-2890. Currently, decisions to use anti-platelet treatments are based on clinical grounds due to the lack of acceptable methods to monitor platelet function in individual patients. The main problem with monitoring platelet function in patients is the artificial in vitro activation of platelets that occurs in conventional techniques. Several instruments have been developed to assess the global platelet response to anti-platelet therapy. A well-established method to assess platelet activation has been studied in whole blood using fluorescence flow cytometry. Michaelson, A. D. Blood Coagul. Fibrinolys. (1994) 5:121-131. The technique is specific and sensitive, since fluorescence measurements can detect as few as 0.8% activated cells. However, the technique is expensive, labor-intensive, and data analysis is imprecise. Further, in some patients, activated platelets lose their marker receptors but continue to circulate and function, which leads to missed detection with activation-specific fluorescence-labeled antibodies Michaelson, A. D. et al., Methods (2000) 21:259-270.
As a high level of platelet function inhibition has been reported to be associated with a decrease in the incidence of major adverse cardiac events, the question becomes whether clinicians should uniformly use aggressive and costly anti-platelet strategies without individual assessment of platelet status in patients with platelet-affected disorders. A simple and rapid method of early detection of platelet activation status without artificial activation of platelets would therefore be useful in identifying those particular patients who would benefit from platelet antagonist therapy, as distinguished from those in whom a material response to anti-platelet medicaments is not foreseen. Further, a simple, reliable and rapid method of monitoring treatment would be useful in optimizing safety and efficacy of anti-platelet therapy.
Platelets not only play an important role in the coagulation system, but are also an integral part of the inflammatory response. Shebuski R. J., Kilgore K. S. J. Pharmacol. Exp. Therapeut. (2002) 300:729-735; Aukrust P. et al., Heart (2001) 86:605-606; Freedman J. E. and J. Loscalzo. Circulation (2002) 105:2130-2132. The contribution of inflammation to the complications of acute ischemic syndromes and coronary revascularization has been increasingly recognized in recent years. Inflammation plays a pivotal role in vascular injury and repair. Accordingly, the inflammatory marker C-reactive protein (CRP) has emerged as a powerful predictor for cardiovascular disease, associated with future cardiovascular events in seemingly healthy subjects and with worse prognosis in acute coronary patient. Lagrand W. K. et al., Circulation (1999) 100:96-102; Ridker P. M. et al., N. Engl. J. Med. (2002) 347:1557-1565; Ridker P. M. et al., N. Engl. J. Med. (2000) 342:836-843; Ridker P. M. et al., Circulation (2000) 101:1767-1772.
Elevated circulating inflammatory markers, in particular CRP, have been associated with elevated risk for both short-term and long-term ischemic events among patients with unstable angina (Lindahl B. et al., N. Engl. J. Med. (2000)343:1139-1147; Liuzzo G. et al., N. Engl. J. Med. (1994) 331:417-424), myocardial infarction (Tommasi S. et al., Am. J. Cardiol. (1999) 83:1595-1599), or those undergoing percutaneous angioplasty (Buffon A. et al., J. Am. Coll. Cardiol. (1999) 34:1512-1521; Versaci F. et al., Am. J. Cardiol. (2000) 85:92-95; Chew D. P. et al., Circulation (2001) 104(9):992-997). Elevated pre-angioplasty CRP levels predicted an increased rate of procedural and in-hospital complications (Buffon A. et al., J. Am. Coll. Cardiol. (1999) 34:1512-1521) an increased risk of death or MI at 1 month follow-up (Chew D. P. et al., Circulation (2001) 104(9):992-997), and 6 months follow-up (Heeschen C. et al., J. Am. Coll. Cardiol. (2000) 35:1535-1542) compared with those patients who have a normal CRP. At 1-year follow-up, clinical restenosis developed in 63% of patients with high pre-angioplasty CRP levels versus only 27% of those with values in the reference range. In fact, elevation of CRP before early revascularization for non-ST-elevation ACS predicts mortality for up to 5 years. Mueller C. et al., Circulation (2002) 105; 1412-1415.
The association between platelet activation and markers of inflammation has been reported in different pathologies. In general, inflammation can promote thrombus formation and can enhance clot stability. The relationship between systemic infection or inflammation and an increased risk of thrombotic diseases has recently raised renewed interest. In patients with acute thrombotic stroke, Toghi et al. showed that platelet aggregation induced by ADP was significantly higher where CRP levels were elevated compared to control donors with normal CRP levels. The data suggest that platelet function may be, in part, related to stroke onset in patients with increased CRP levels. Toghi H. et al., Thromb. Res. (2000) 100:373-379. In hypercholesterolemia, in vivo platelet activation measured by soluble P-selectin and urinary 11-debydro-TxB2 excretion, are associated with increased sCD40L levels (Cipollone F. et al., Circulation (2002) 106:399-402) and IL-1β and CRP (Ferroni P et al., Circulation (2003) 10814:1673-1675), markers of systemic inflammation. In obese women, increased platelet activation was driven by high CRP and both were reversible with loss of weight (Davi G. et al., JAMA (2002) 288:2008-2014).
These inflammatory processes may be reciprocally affected by platelet activation. Toghi H. et al., Thromb. Res. (2000) 100:373-379; Neumann F.-J. et al., Circulation (1997) 95:2387-2394. Platelet activation can increase inflammation through several mechanisms. Platelets release a wide range of growth factors and inflammatory mediators from intracellular storage organelles. Products of activation may aid neutrophil accumulation and enhance inflammation. Activated leukocytes and platelets potentiate each others' effects. The important mediators released by platelets are P-selectin (CD62p), CD40p, platelet activating factor, macrophage chemotactic factor-1, interleukin-1, thrombospondin and fibronectin. These mediators are rapidly expressed by activated platelets. Weyrich A. S. et al., J. Clin. Invest. (1996) 97:1525-1534; Carlos T. M. et al., Blood (1994) 84:2069-207; Zimmerman G. A. et al., Crit. Care Med. (2002) 30:294-301; Valles J et al., Blood (2002) 99:3978-3984; Sarma J. et al., Circulation (2002) 105:2166-2171; O'Brien K. D. et al., Circulation (1996) 93:672-682. Davi et al. reported a biochemical mechanism suggesting that a low-grade inflammatory state may be the primary trigger of thromboxane-dependent platelet activation mediated through enhanced lipid peroxidation. Davi G. et al., JAMA (2002) 288:2008-2014. Other mechanisms, such as a direct proinflammatory effect of CRP, are likely to amplify and sustain the relationship between systematic inflammation and platelet activation. Pasceri V. et al., Circulation (2000) 102:2165-2168.
Increased level of basal platelet activity has been correlated with adverse events. Stemhubl S. R. et al., Circulation (2001) 103:2572-2578. It is hypothesized that the baseline platelet activation status may be predictive of future platelet activity accompanied by vascular complications. Gurbel P. A. et al., Thrombosis Res. (2000) 99:105-107; Gurbel P. A. et al., Scand. Cardiovasc. J. (2000) 34:53-58. There is an urgent need for identifying patients who may be susceptible to future untoward platelet events associated with vascular complications so that these patients can be earmarked for more aggressive anti-platelet regimens. Indeed, Ridker et al. showed in the Physician's Health Study that patients at the highest level of risk, as measured by CRP, derive the most benefit from effective anti-platelet therapy. See Ridker P. M. et al., N. Engl. J. Med. (1997) 336:973-979.
There is a need in the art for a rapid, simple test for platelet activation that can be used in a dynamic fashion to detect platelet activation and predict vascular problems as well as to monitor platelet activation in patients undergoing anti-platelet therapy and/or angioplasty.